Tumor stem cells are a subpopulation of cells isolated from tumors, including glioblastoma, that have stem-like properties and the ability to efficiently initiate tumor formation. Glioblastoma is a primary malignant brain tumor that has remained largely refractory to all forms of therapy and is almost uniformly lethal with a median survival of approximately 15 months. There is increasing evidence that tumor stem cells play an important role in glioblastoma progression, resistance to treatment, and ultimate recurrence. In contrast to the bulk tumor, glioblastoma stem cells (GSCs) have the ability to self-renew, differentiate into more mature and less tumorigenic phenotypes, and give rise to the heterogeneous cell types that are a hallmark of glioblastoma. We have isolated and characterized a panel of GSC lines, and these cells form tumors that recapitulate the histopathology and heterogeneity observed in the patients' tumors from which they were isolated. Studies of tumor stem cells have thus far been based on our understanding of normal stem cells and development. However, addressing fundamental questions about how tumor stem cells are distinct from normal stem cells, and how these differences can be exploited for therapeutic benefit are likely to require different approaches. Thus, we are proposing to discover compounds that induce differentiation of GSCs, but not neural stem cells, in unbiased high throughput screens of FDA approved and known bioactives, an approach that has not been previously described. The validated 'hits' should shed light on cellular pathways selectively involved in the maintenance and differentiation of GSCs, and because GSC differentiation impairs tumorigenicity, lead to the development of novel drugs targeting GSCs for the treatment of glioblastoma. To accomplish this, we will develop two assays to screen for differentiation inducers: (1) a high-content image-based immunofluorescence assay of endogenous markers of stemness and differentiation in adherent GSC cultures, and (2) a luminescence-based assay of promoter-driven luciferase reporters in transduced GSCs in sphere culture. The GSC reporter lines (from (2)) provide a model to non-invasively follow GSC differentiation during tumor progression in vivo in real time. These approaches are directly translatable to tumor stem cells from other types of cancer and should provide tools and reagents that will benefit the field and patients with glioblastoma.